!CANCER RESEARCH 56, 2468-2471.
June 1. 9961
Advances in Brief
Glucose Catabolism in Cancer Cells: Amplification
of the Gene Encoding
Type II Hexokinase1
Annette
Rempel,
Saroj P. Mathupala,
Constance
A. Griffin,
Anita L. Hawkins,
and Peter L Pedersen2
Departments of Biological Chemistry [A. R.. S. P. M., P. L P.] and Pathology [C. A. G.. A. L H.], The Johns Hopkins University, School of Medicine, Baltimore, Maryland
21205-2185
Abstract
Hexokinase type II is highly overexpressed in many cancer cells, where
it plays a pivotal role in the high glycolytic phenotype. Here we demon
strate
by Southern
blot analysis
and fluorescence
in situ hybridization
(FISH) that in the rapidly growing rat AS-30D hepatoma cell line, en
hanced hexokinase
activity is associated
with at least a 5-fold amplification
of the type II gene relativeto normalhepatocytes.This amplificationis
located chromosomally, extends to the whole gene, and most likely occurs
at the site of the residentgene. No rearrangementof the gene could be
detected.
Therefore,
overexpression
of hexokinase
type II in AS-30D
hepatoma cells may be based, at least in part, on a stable gene amplifica
tion. This is the first report describing the amplification of a hexokinase
regulation of the tumor HKII3 gene (4) by elucidating the sequence of
its promoter region and identifying activators thereof. The present
paper focuses on structural differences between the HKII gene in
normal and tumor cells as a possible mechanism for gene induction.
For our studies, we used the highly glycolytic, rapidly growing rat
hepatoma cell line AS-30D. This cell line has been characterized in
detail in this laboratory with respect to its high glycolysis and the role
in this process of hexokinase (Refs. 2, 10, and references therein). The
data described below indicate that the hexokinase gene is amplified at
least 5-fold in AS-30D hepatoma cells relative to normal hepatocytes,
and that no rearrangement of the gene occurs.
gene in a tumorcell line expressingthe high glycolyticphenotype.
Materials
Introduction
and Methods
Cells and Cell Culture.
One of the most common and profound phenotypes of malignant
tissues, particularly those with the highest growth rates, is their
capacity to utilize and catabolize glucose at high rates (1). The high
glycolytic rate is important for rapidly proliferating cancers not only
as a major energy source, but also to provide such cells with precur
sors for nucleotide and lipid biosynthesis. Altered expression of
glycolytic enzymes, especially hexokinase (EC 2.7.1.1) is believed to
play a major role in this phenomenon (1—3).Hexokinase catalyzes the
conversion of glucose to glucose-6-phosphate in the first step of the
glycolytic pathway. Hexokinase activity (1—3),mRNA level (3, 4),
and transcription rate (5) are increased markedly in rapidly growing
tumors. To further potentiate the enhanced hexokinase activity
achieved by overexpression, most of the enzyme is bound to the outer
mitochondrial membrane, where it has direct access to mitochondri
ally generated AlP and is less sensitive to glucose-6-phosphate inhi
Clone 9 (CRL
1439),
a rat hepatocyte
cell line,
was obtained from the American Type Culture Collection and grown in RPM!
1640 medium. AS-30D hepatoma cells were grown in the peritoneal cavity of
female Sprague-Dawley rats, harvested, and purified as described previously
(4). Hepatocytes were isolated from female Sprague-Dawley
collagenase perfusion method (12).
rats by the
Hexokinase Assay. Hexokinase activity was determined spectrophoto
metrically
on whole-cell
lysates
using a glucose-6-phosphate
dehydrogenase
coupled assay (6). Activity is expressed in mUs, I mU defined as the formation
of 1 nmol NADPH/min.
Southern Blot Analysis. High molecularweight DNA was isolated from
AS-30D hepatoma cells and hepatocytes as described (13). DNA (30 p.g) was
digested with the indicated restriction enzymes. To avoid technical problems
resulting from incomplete hydrolysis, digestions were repeated several times
with an excess
of restriction
enzymes.
The digested
DNA was fractionated
on
bition (2, 6). In brain tumors, hexokinase activity is proportional to the
a 1% agarose gel and transferred to nylon membranes (Amersham). Probe
labeling, hybridization, and detection were performed with the Fluorescein
Gene Images System (Amersham) according to the manufacturer's instruc
degree of malignancy (7). In addition, Fanciulli et a!. (8) demonstrated
that increased hexokinase activity may not only be the consequence of
tions. Either the full-length cDNA or a 260-bp fragment corresponding to the
position —197 to +63 of rat skeletal muscle HKII (11) were used as probes.
altered metabolic requirements of cancer cells but may also be a
FISH. The pUC18 plasmid containingthe 3.6-kb cDNA clone of the rat
HKII gene was nick translated with biotin-14 dATP (Bethesda Research
Laboratories, Gaithersburg, MD), with 25% incorporation as determined by
modification per se to increase mitotic activity. Therefore, elucidation
of the molecular basis underlying hexokinase overexpression will
provide information that is not only useful in explaining the mecha
nism of the high glycolytic phenotype but may lead also to new
approaches in cancer diagnosis and therapy. Of the four known
hexokinase isozymes (I, II, III, and IV), it is the type H, and to a lesser
extent the type I isozymes that are overexpressed in rapidly growing,
highly glycolytic tumors examined to date (3, 4, 9—11). In a recent
study, we addressed for the first time the issue of transcriptional
tritium tracer incorporation.
Slides with chromosome spreads were made from
AS-30D hepatoma cells and clone 9 (normal control), harvested by standard
cytogenetic techniques. FISH was performed
cations. Probe mix [2X SSCP (1 X SSCP =
citrate, 0.02 M sodium phosphate, pH 6.0),
sulfate, 5 nW@l biotinylated probe, and 20
as described (14) with modifi
0. 15 M NaCl, 0.015 M sodium
50% formamide, 10% dextran
@g4dsalmon sperm DNAI was
denatured at 70°Cfor 5 mm, chilled quickly on ice, placed on slides, and
hybridized at 37°Covernight. Slides were washed in 50% formamide and 2X
SSC(1X SSC = 0.15MNaC1,0.015Msodiumcitrate,pH 7.0)at 43°C
for 20
Received 3/1 2/96; accepted 4/23/96.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
mm, and two changes of 2X SSC at 37°Cfor 5 mm each. Biotinylated probe
18 U.S.C. Section 1734 solely to indicate this fact.
using reagents
I Supported
in
part
by
NIH
Grant
CA
32742
(to
P.
L.
P.)
and
NIH
Grant
2P30-
CA06972 (to C. A. G.). A. R. was an awardee of the Deutsche Forschungsgemeinschaft.
2 To
whom
correspondence
should
be addressed,
at Department
of Biological
was detected with FITC-avidin
with biotinylated
Kit (Oncor
antiavidin,
Inc., Gaithersburg,
MD), following manufacturer's instructions.
Chem
istry, The Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore,
MD 21205-2185.Phone:(410)955-3827;Fax:(410)955-1944.
and amplified
from an In Situ Hybridization
3 The
abbreviations
used
are:
HKII,
hexokinase
type
II;
FISH,
fluorescence
hybridization; mU, milliunit.
2468
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in situ
GENE AMPLIFICATION OF TYPE ll HEXOK!NASE IN TUMOR CELLS
from different Southern blots confirmed the data obtained in the
Results and Discussion
dilution experiment, and a factor of approximately 5 was calculated
for the amplification. Additional support for the ilK!! gene amplifi
cation in AS-30D hepatoma cells came from experiments searching
Preliminary Southern blot analysis using digested genomic DNA
from hepatocytes and AS-30D hepatoma cells revealed that the HKII
probe hybridized with much greater intensity to the hepatoma DNA
than to the hepatocyte DNA. To estimate the differences in hybrid
ization intensities we performed a dilution experiment. The hybrid
ization signals with different amounts of EcoRI- and XbaI-digested
AS-30D hepatoma genomic DNA were compared to the signal ob
mined with 30 @gof DNA isolated from hepatocytes (Fig. 1). The
for the HKH promoter region in these cells and in hepatocytes. Thus,
six positive plaques were obtained when 5 X lO@plaques were
screened from an AS-30D hepatoma genomic library, whereas only
two positives were found in 2.5 X 106 plaques of a normal liver
library. Taking into consideration that the liver library had been
amplified previously, the estimated factor for amplification is near 6,
in accordance with the results from Southern blot analysis.
Instability of the genome is a well-known phenomenon of trans
formed cells and amplification is a frequently observed mechanism
for the overexpression of oncogenes, including N-myc (15) and the
epidermal growth factor receptor gene (16). It is well known that a
strong relationship exists frequently between a gene that is amplified
blots were probed with two different probes specific for the HKIJ gene
(Fig. 1, A and B). The intensities of the resulting bands indicate that
3—6 @ghepatoma DNA were equivalent to 30 ,.@ghepatocyte DNA.
From this experiment, we estimated that AS-30D hepatoma cells
contain approximately 5—10-foldmore copies of the HKII gene than
normal hepatocytes. In addition, it is clear from Fig. lA that the signal
intensities of all type II hexokinase-related bands obtained with AS
and cell growth. The amplification of the HKII gene is consistent with
30D hepatoma DNA are the same. This indicates that the amplifica
this relationship,becausethe role of this criticalmetabolicenzymeis
tion extends to the whole coding region of the hexokinase gene.
Moreover, when the membranes were probed again with DNA frag
ments specific for the 5'-flanking region of the hexokinase gene (4),
to provide cells with both energy and precursors for nucleotide and
lipid biosynthesis. In a recent report (4), we provided evidence that
increased expression of one or more transcription factors is involved
similar results were obtained (data not shown). Thus, the amplified
in the elevated production of HKH in AS-30D hepatoma cells. Work
unit in AS-30D hepatoma cells also includes the promoter region of
presented here suggests that amplification of the gene for the same
the HKII gene. Densitometric quantification of autoradiograms made
enzyme may play a role as well.
Southern blot analysis (Fig. 1A) displayed some faint restriction
A
a
b
c
d
e
f
fragments with the hepatocyte DNA that were not observed in the
AS-30D hepatoma DNA. As the restriction enzymes used, EcoRI and
XbaI, are both sensitive to methylation of their recognition sequence,
Cab
this raisesthe possibilitythat methylationdifferencesexist withinthe
kbp
HKJI gene in normal hepatocytes and AS-30D hepatoma cells. Several
studies reviewed in Ref. 17 have demonstrated that DNA methylation
plays a role in gene regulation. Therefore, methylation could be
9.4 —
6.6 —
involved in differential expression of HKII in normal and tumor cells.
Additional experiments to test this hypothesis are in progress.
For some oncogenes, it is well known that amplification is accom
panied by recombination and rearrangement of the gene locus (18). To
4.4 —
look for structural
differences
in the HKII gene locus in normal
and
AS-30D hepatoma cells, RFLP analysis was carried out. To circum
vent problems due to methylation differences of normal and tumor
DNA, methylation-insensitive restriction enzymes (RsaI, NdeI,
HindllI) were used. For each enzyme, the same restriction fragment
2.3
2.0 —
pattern is observed in both hepatocyte and AS-30D hepatoma DNA
(Fig. 2). Thus, no macroscopic rearrangement of the hexokinase gene
is seen at this level of resolution. Also, this result renders it unlikely
that a translocation of the hexokinase gene locus has occurred in
AS-30D hepatoma cells. Therefore, the amplification described above
appearsto occurat the siteof theresidentgene,andthe possibilitythat
S.
B
the HKII gene in AS-30D hepatoma cells has come under the control
of different regulatory sequences through translocation seems remote.
To obtain additional support for the amplification and localization
of the HKJJ gene, in situ hybridization experiments were performed.
Because primary hepatocytes divide very rarely and dedifferentiate
rapidly, we used clone 9 (CRL 1439), a nontumorigenic, normal liver
9.4
6.6
Fig. 1. Amplificationof the HKIIgenomic sequencein AS-30D hepatomacells. High
molecular weight genomic DNA was digested with EcoPJ and XbaI. Hepatocyte DNA, 30
@ag
(Lane a); AS-30D hepatoma DNA, 30 ,sg (Lane b); and serial dilutions of the AS-30D
hepatoma DNA (Lanes c—f,
containing, respectively, 12, 6, 3, and 1.5 gagof DNA) were
sizefractionatedon a 1%agarosegelandtransferredto a nylonmembrane.Theblotwas
hybridizedto a HKHfull-lengtheDNA (A) or to a 26O-bpfragmentcorrespondingto the
position —
197 to +63 of HKH(B). The blot was strippedof signal between hybridiza
tions. Molecular weight markers(A-Hindffl) are shown to the left. Equal loading of
hepatocyteand undilutedAS-30D hepatomaDNA was estimatedby ethidiumbromide
staining of the gel (C).
cell line (19) as a control for in situ hybridization. As shown in Table
1, these cells exhibit no detectable hexokinase activity, in contrast to
AS-30D hepatoma cells where the activity is 762 mU/mg. The liver
homogenate, which in addition to hepatocytes contains other cell
types, exhibits a low but detectable hexokinase activity. In situ hy
bridizations (Fig. 3) using the HKII cDNA as probe revealed that in
AS-30D hepatoma cells, a signal could be detected readily in every
(20/20)metaphase
andinterphase
cell.Occasional
(4/20)tetraploid
cells that were observed in the AS-30D hepatoma cell population
showed a hybridization signal on two chromosomes, indicating that
2469
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GENE AMPLIFICATION OF TYPE II HEXOIUNASE IN TUMOR CELLS
abc
d
f
e
kbp
abundant in AS-30D hepatoma cells than in control cells. Although
FISH
does
notallow
exact
quantitation
oftheamplification,
itis
consistent with at least a 5-fold increase in copy number. Moreover,
the amplified sequence was localized to a single chromosome in
AS-30Dhepatoma
cells,suggesting
thattheamplificationis present
on only one of the two homologous chromosomes, a finding not
uncommon for amplified genes (20). Chromosomally localized gene
amplification represents one of the more stable fonns of amplified
genes. Stable retention of amplified genes and their passage to daugh
tar progeny are ensured only when such genes are integrated within a
chromosome. Unstable amplified genes that are very common in
transformed cells are associated characteristically with extrachromo
somal elements called double minutes. However, double minutes were
never observed in our studies of AS-30 hepatoma cells.
In summary, results reported here provide for the first time cvi
9.4-6.6-4.4--
@11
—
I,.
A.
2.3 -.
2.0-.4
@
t 1I@!L@c:@
@
@
f
.
\
Fig. 2. RFLPanalysisofliver hepatocyteandAS-30D hepatomaDNA. GenomicDNA
isolated from AS-SOD cells [10 ,.@g](Lanes a, c, and e) and hepatocytes [30 sag] (Lanes
b, d@andI) was digestedto completion with methylation-insensitiverestrictionenzymes
(Lanes a and b, RsaI; Lanes c and d@NdeI; Lanes e and f Hincffl), separated by gel
electrophoresis, and transferred to a Hybond filter. The blot was hybridized to a fluores
cein-labeled full-length HKII cDNA and developed by using an antifluorescein alkaline
phosphatase conjugate and chemiluminescence.
Hindffl-digested
A-DNA was used as a
marker. No differences between normal and tumor DNA were detected.
Table 1 Hexokinase activity in normal
AS-30Dhepatoma
rat liver, hepatocytes (clone 9), and
cellsHexokinase
B.
“Materialsand
activity in whole-cell lysates was determined as described in
Methods.―
SD.Cell
Values representthe mean of multipledatenninations±
(mU/mg)Normalratliver
source
1.2Hepatocytes
9)AS-30D (clone
52a
hepatoma
@
Hexokinaseactivity
12±
762 ±
not detectable.
the gene was amplified before the chromosomes were duplicated. The
single positive chromosome seen in the AS-30D sample most likely
represents the amplification site on one chromosome homologue only,
but the loss of the other homologous chromosome cannot be ruled out.
In contrast, in clone 9, no interphase signals were seen, and only 1 of
20 metaphase cells showed a faint specific signal. Because the probe
used for in situ experiments was rather small (3.6 kb), genes with a
low copy number cannot be detected easily with this size probe. This
confirms again that the copy number of the HKtI gene is much more
Fig. 3. in situ hybridization.The biotin-labeledprobe (pUC18, containingthe 111(11
cDNA) was hybridizedto metaphaseand interphasechromosomesfollowed by fluores
cein immunodetection.A single block offluorescent signal was detectedeasily on a single
chromosome of AS-30D hepatoma cells (A), whereas no signal was observed on the
hepatocyte (clone 9) chromosomes
(B).
2470
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GENE AMPLIFICATIONOF TYPE IIHEXOKINASE IN TUMOR CELLS
dence that a hexokinase gene (type II) is amplified in a tumor cell line
exhibiting a high glucose catabolic phenotype. This amplification is
stable, not associated with a rearrangement
of the hexokinase gene
locus, and occurs probably at the site of the resident gene.
10. Nakashima, R., Paggi, M. G., Scott, L. J., and Pedersen, P. L. Purification and
characterization
of a bindable form of mitochondrial
bound hexokinase
from the
highly glycolytic AS-30D rat hepatoma cell line. Cancer Res., 48: 913—919,1988.
11. Thelen, T. A., and Wilson, J. E. Complete amino acid sequences of the type II
isozyme of rat hexokinase, deduced from the cloned cDNA: comparison with a
hexokinase from Novikoff ascites tumor. Arch. Biochem. Biophys.. 286: 645—651,
1991.
12. Freshney, R. I. Culture of Animal Cells: A Manual of Basic Technique, Ed. 2, pp.
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Glucose Catabolism in Cancer Cells: Amplification of the Gene
Encoding Type II Hexokinase
Annette Rempel, Saroj P. Mathupala, Constance A. Griffin, et al.
Cancer Res 1996;56:2468-2471.
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